دورية أكاديمية
Real-Time Visualization of 'in Vitro' Transcription of a Fluorescent RNA Aptamer: An Experiment for the Upper-Division Undergraduate or First Year Graduate Laboratory
| العنوان: | Real-Time Visualization of 'in Vitro' Transcription of a Fluorescent RNA Aptamer: An Experiment for the Upper-Division Undergraduate or First Year Graduate Laboratory |
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| اللغة: | English |
| المؤلفون: | Heili, Joseph M., Gomez-Garcia, Jose, Gaut, Nathaniel J., Cash, Brock W., Aufdembrink, Lauren M., Heffron, Brent A., Shirley, Joshua D., Carlson, Erin E. (ORCID |
| المصدر: | Journal of Chemical Education. Oct 2018 95(10):1867-1871. |
| الإتاحة: | Division of Chemical Education, Inc. and ACS Publications Division of the American Chemical Society. 1155 Sixteenth Street NW, Washington, DC 20036. Tel: 800-227-5558; Tel: 202-872-4600; e-mail: eic@jce.acs.org; Web site: http://pubs.acs.org/jchemeduc |
| Peer Reviewed: | Y |
| Page Count: | 5 |
| تاريخ النشر: | 2018 |
| نوع الوثيقة: | Journal Articles Reports - Descriptive |
| Education Level: | Higher Education Postsecondary Education |
| Descriptors: | Science Instruction, Laboratory Experiments, Genetics, Undergraduate Students, Graduate Students, College Science, Science Laboratories, Biochemistry, Visualization |
| DOI: | 10.1021/acs.jchemed.7b00735 |
| تدمد: | 0021-9584 |
| مستخلص: | A teaching laboratory experiment is described where students prepare in vitro transcription reactions of a fluorescent RNA aptamer, named Broccoli, and observe the production of the aptamer in real-time on a fluorescence plate reader. Alternate visualization methods with minimal costs are also described for laboratories lacking this instrumentation. Two optional experiments are also described. Optional Experiment 1 involves purification of RNA transcription reactions using a commercial spin column kit and having students correlate cleanup kit yield with transcribed aptamer fluorescence. Optional Experiment 2 involves running a polyacrylamide gel of the transcription reaction with a ladder, followed by staining with (Z)-4-(3',5'-difluoro-4'- hydroxybenzylidene)-2-methyl-1-(2?,2?,2?-trifluoroethyl)-1H-imidazol-5-(4H)-one (DFHBI-1T) (selective for Broccoli) and a second stain with SYBR Gold (nonselective, allowing for simultaneous visualization of Broccoli and ladder). This experiment has the practical advantage of enabling aptamer visualization in laboratories without a fluorescence spectrometer or plate reader, as well as the pedagogical benefit of demonstrating specific activation of the fluorescence of a small molecule by an RNA aptamer in another context (gel staining). Each experiment allows students to perform straightforward, easily understood teaching laboratory experiments, including key concepts in cellular imaging, and RNA biochemistry widely employed in biochemical research. |
| Abstractor: | As Provided |
| Entry Date: | 2018 |
| رقم الأكسشن: | EJ1195678 |
| قاعدة البيانات: | ERIC |
Find this issue from the American Chemical Society | تدمد: | 0021-9584 |
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| DOI: | 10.1021/acs.jchemed.7b00735 |